Engineered galactose oxidase variant enzymes

Abstract

The present invention provides engineered galactose oxidase (GOase) enzymes, polypeptides having GOase activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Methods for producing GOase enzymes are also provided. The present invention further provides compositions comprising the GOase enzymes and methods of using the engineered GOase enzymes. The present invention finds particular use in the production of pharmaceutical and other compounds.

1 . An engineered galactose oxidase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 2, 4, 166, 272, 928, 932, 1264, 1416, 1598, 1866, 1912, 2080, 2300, and/or 2424, or a functional fragment thereof, wherein said engineered galactose oxidase comprises at least one substitution or substitution set in said polypeptide sequence, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2, 4, 166, 272, 928, 932, 1264, 1416, 1598, 1866, 1912, 2080, 2300, and/or 2424, and wherein said engineered galactose oxidase comprises at least one improved property compared to wild-type  F. graminearum  galactose oxidase. 
     
     
         2 . An engineered galactose oxidase comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NOS: 2, 4, 166, 272, 928, 932, 1264, 1416, 1598, 1866, 1912, 2080, 2300, and/or 2424, or a functional fragment thereof. 
     
     
         3 . The engineered galactose oxidase of  claim 1 , wherein the polypeptide sequence of the engineered galactose oxidase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:1866, and wherein said engineered galactose oxidase comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 29/149/463/601, 29/177/197/592, 29/177/463, 29/197/592, 29/463, 62/208/417/615, 62/286/615, 62/373/466, 62/466, 62/466/597, 149, 149/208/615, 149/463, 177/194/197/463/565, 177/197/463/565, 177/280/463/594/601, 177/463/565, 177/463/592, 184, 197, 197/280/463, 197/463/592, 197/466/569/596, 208/251/259/278, 234, 234/384, 251, 251/399/615, 278, 373/466, 384/569, 399/615, 417/615, 463/565, 466, 546, 569, and 569/597, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2. 
     
     
         4 . The engineered galactose oxidase of  claim 1 , wherein the engineered galactose oxidase comprises production of 2-ethynylglyceraldehyde with increased enantiomeric excess of the R-stereoisomer as compared to wild-type  F. graminearum  galactose oxidase. 
     
     
         5 . The engineered galactose oxidase of  claim 1 , wherein the polypeptide sequence of the engineered galactose oxidase has at least 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 4, and wherein said engineered galactose oxidase comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 170, 171, 173, 191, 192, 193, 194, 197, 198, 199, 202, 204, 205, 220, 227, 243, 247, 248, 252, 269, 294, 296, 324, 332, 407, 463, 465, 466, 493, 515, 517, 520, 521, and 522, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 4. 
     
     
         6 . The engineered galactose oxidase of  claim 1 , wherein the polypeptide sequence of the engineered galactose oxidase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:1866, and wherein said engineered galactose oxidase comprises at least one substitution selected from positions 197, 463 and 592 in said polypeptide sequence wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 1866. 
     
     
         7 . The engineered galactose oxidase of  claim 1 , wherein the polypeptide sequence of the engineered galactose oxidase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 1866, and wherein said engineered galactose oxidase comprises at least one substitution selected from positions 197P, 463V and 592G in said polypeptide sequence wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 1866. 
     
     
         8 . The engineered galactose oxidase of  claim 1 , wherein the polypeptide sequence of the engineered galactose oxidase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 1866, and wherein said engineered galactose oxidase further comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 29, 29/30/50/79/136/197, 29/30/50/79/197/407, 29/30/79/136/156/197, 29/30/79/197, 29/30/79/197/407, 29/30/136/197/407/486, 29/30/136/407, 29/30/197, 29/30/197/407, 29/50/197/407/486, 29/197/407, 29/197/407/486, 30, 30/50/79/136/156/197, 43/197/407, 50/136/197/486, 65, 79, 79/136/197/407, 79/156/197/407, 136, 136/197/407, 136/197/486, 156/161/486, 197, 197/407, 197/486, 279/291/375/420/429/436/453/465, 279/291/375/465/536/538, 279/291/465, 279/291/465/536, 279/375/420/429/453/465/472/538, 279/375/420/465, 279/465, 291/375/420/430/465/538, 291/375/436/465/538, 291/375/453/465, 291/420/465/481/538, 291/429/465, 291/453/465/536/538, 291/465, 291/465/538, 375/420/465, 375/429/453/465, 375/465, 420/436/465, 429/465, 453/465, 453/465/478/481, 465, 465/536/538, 486, and 615, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 1866. 
     
     
         9 . The engineered galactose oxidase of  claim 1 , wherein the polypeptide sequence of the engineered galactose oxidase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 1866, and wherein said engineered galactose oxidase further comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 29V/149N/463V/601L, 29V/177L/197P/592G, 29V/177L/463V, 29V/197P/592G, 29V/463V, 62D/466V, 62D/466V/597A, 62G/208F/417L/615I, 62G/286C/615I, 62Q/373D/466V, 62Q/466V, 62Q/466V/597A, 149N/463V, 149R, 149R/208F/615I, 177L/194Q/197P/463V/565S, 177L/197P/463V/565S, 177L/280N/463V/594M/601L, 177L/463V/565S, 177L/463V/592G, 184L, 197A, 197A/466V/569L/596S, 197P/280N/463V, 197P/463V/592G, 208F/251V/259N/278L, 234L, 234L/384N, 251V, 251V/399V/615I, 278L, 373D/466V, 384N/569L, 399V/615I, 417L/615I, 463V/565S, 466V, 546E, 569L, and 569L/597A, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 1866. 
     
     
         10 . The engineered galactose oxidase of  claim 1 , wherein the polypeptide sequence of the engineered galactose oxidase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 1866, and wherein said engineered galactose oxidase comprises the substitution set 197/463/592 in said polypeptide sequence wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 1866. 
     
     
         11 . The engineered galactose oxidase of  claim 1 , wherein the polypeptide sequence of the engineered galactose oxidase has at least 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 2080, and wherein said engineered galactose oxidase further comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 24, 47, 63, 78, 95, 119, 121, 197, 207, 214, 219, 220, 249, 294, 324, 365, 408, 414, 437, 480, 485, 520, 556, 571, 598, 600, and 626, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2080. 
     
     
         12 . The engineered galactose oxidase of  claim 1 , wherein the polypeptide sequence of the engineered galactose oxidase has at least 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 2080, and wherein said engineered galactose oxidase further comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 14/130/257/472, 14/257, 24, 29, 29/136, 29/136/197/436, 29/136/436, 29/136/436/453, 29/197, 29/197/342/436, 29/197/436, 29/197/436/453, 29/197/453, 29/436, 29/436/472, 29/453, 29/472, 43, 63, 95, 119, 130/421, 136, 136/197/436, 136/197/436/453, 136/436, 144, 197, 197/436, 197/436/453, 197/436/472, 197/453, 214, 219, 249, 257, 257/472, 297, 359, 436, 437, 460, 485, 495, 520, 556, 560, 567, and 592, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2080. 
     
     
         13 . The engineered galactose oxidase of  claim 1 , wherein the polypeptide sequence of the engineered galactose oxidase comprises an even-numbered SEQ ID NO set forth in SEQ ID NOS: 1866-2860. 
     
     
         14 . A polynucleotide sequence encoding an engineered galactose oxidase of  claim 1 . 
     
     
         15 . An expression vector comprising at least one polynucleotide sequence of  claim 14 . 
     
     
         16 . A host cell comprising at least one polynucleotide sequence of  claim 15 . 
     
     
         17 . A method of producing an engineered galactose oxidase in a host cell, comprising culturing the host cell of  claim 16 , under suitable conditions, such that at least one engineered galactose oxidase is produced. 
     
     
         18 . The method of  claim 17 , further comprising recovering at least one engineered galactose oxidase from the culture and/or host cell. 
     
     
         19 . The method of  claim 17 , further comprising the step of purifying said at least one engineered galactose oxidase. 
     
     
         20 . A polynucleotide sequence encoding at least one engineered galactose oxidase, wherein said polynucleotide sequence comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NOS: 1, 3, 165, 271, 927, 931, 1263, 1415, 1597, 1865, 1911, 2079, 2299, and/or 2423, wherein the polynucleotide sequence of said engineered galactose oxidase comprises at least one substitution at one or more positions.